1. Field of the Invention
The present invention relates generally to preparations of stem cells, particularly cancer stem cells derived from neural crest tissue. The invention also relates to methods of isolating cancer stem cells, and various methods for using cancer stem cells in diagnostic, therapeutic and other clinical and non-clinical applications.
2. Related Art
Neuroblastoma (NB) is the most common extracranial solid tumors in children, with poor survival rates in children with metastatic disease. NB is estimated to be responsible for about 15% of cancer-related deaths in children (1, 2). The survival rate for metastatic NB is estimated to be less than 30%. In the majority of these cases, conventional cancer therapies have been ineffective.
Little is reported concerning the precise molecular alterations that give rise to NB, its cell of origin, or why NB cells metastasize and become resistant to chemotherapeutic agents. Unfortunately, genetic mutations that contribute to the origin and progression of 98% of NB cases have not been identified.
One identifiable hallmark of NB is the appearance of proliferating cells with characteristics of neural crest-derived sympathetic neuronal precursors (neuroblasts). NB tumors also frequently contain other neural crest cell types, including neuroendocrine and Schwann cells. Moreover, NB appears in tissues that developmentally derive from the neural crest including sympathoadrenal precursors which differentiate into both sympathetic neurons and adrenal chromaffin cells, the paravertebral and preaortic sympathetic ganglia, and the adrenal gland.
The clinical behavior of NB is unique. Tumors that arise in children under one year of age may spontaneously regress by differentiation or apoptosis, even after arising in or metastasizing to liver and skin. In contrast, NB tumors in children over one-year-old often grow aggressively, disseminate to the bone and bone marrow, and are fatal in the vast majority of cases.
Mass screening of infants showed that NB is much more frequent than previously thought. Many of these tumors regress without clinical diagnosis. Regressing or favorable-prognosis tumors have been reported to express high levels of the TrkA/NGF receptor and display phenotypes of differentiated peripheral neural cells, while malignant or unfavorable-prognosis tumors resemble proliferating sympathoadrenal precursors, often expressing TrkB, amplified MYCN, and many genes involved in neural crest development.
The only reported germline NB predisposition gene is Phox2b, which is mutated in many familial cases of NB, and is required for proper differentiation of sympathetic neurons from neural crest precursors (NCPs) (3,4). In the regressive form of the disease, the precursor cells ultimately differentiate or die, while in older children, these molecular transformations instead result in a population of persistently proliferating and highly migratory transformed neuroblasts.
The concept of tumor-initiating cells (TIC) (also called tumor or cancer stem cells) postulates that only rare cells in tumors are endowed with tumorigenic potential, and was initially developed to explain why (i) most tumors are comprised of both undifferentiated proliferating progenitors and post mitotic differentiated cells, (ii) only a very small fraction of tumor cells form colonies after plating in vitro, and (iii) large numbers of tumor cells are required to seed the growth of a new tumor in mice (4-10).
Dick et al. and others reported that clonally-derived tumor cells of acute myelogenous leukemia (AML) patients could be physically separated into tumorigenic and non-tumorigenic fractions (11, 12). Brain and breast tumors have also been reported to contain a subpopulation of TICs (13, 14). Thus, in solid tumors, a rare tumor cell population may fuel tumor growth and seed metastasis. This hypothesis has major implications for treating cancer patients. For example, many current therapies kill the bulk of proliferating tumor cells, but these cells may not be intrinsically tumorigenic, and in many cases the TICs may escape the effects of the therapeutic agents, leading to tumor relapse. Thus, it is essential to identify and characterize TICs from various tumors in order to develop and target therapies against this critical cell type.
TICs have also been shown to share phenotypic characteristics with stem cells derived from their tissue of origin. For example, for a given tissue, the tissue stem cells and TICs both (i) self-renew, (ii) express common phenotypic markers, (iii) grow in a similar fashion in response to mitogens, and (iv) yield tissue-appropriate progeny (13, 14). However, whereas tissue stem cells generate mature differentiated cell types, differentiation of TICs is generally arrested at the level of one or more tissue progenitor cells resulting in tumors comprising a hierarchy of progenitors and some differentiated progeny (4).
Many pediatric and adult tissues contain resident stem cells (4). It is currently unknown if TICs originate by transformation of tissue stem cells. Observations have been made that oncogenic mutations commonly affect genes required for normal stem cell renewal and differentiation (4). This may be particularly relevant for children's tumors, since developing tissues contain a higher proportion of tissue stem cells than do adult tissues.
Tumor initiating cells from some solid tissue tumors, such as breast and brain tumors, have been described. However, a TIC population from tumor tissue in a patient with NB has not been isolated. One reported observation in some infantile forms of NB (called stage 4S) is that large tumors are frequently found in skin (15). It was previously assumed that skin was a preferred metastatic target for NB. However, a population of TICs from such solid tumor tissue has not yet been reported.
The above and other observations in the field reveal a continuing medical need continues to exist in the art to determine why and in which cell type NB arises, and why some NB tumors spontaneously regress and others are fatal. In addition, new effective drug targets and therapeutics tailored to identifying and treating specific forms and stages of NB are needed.